In earlier work, we found that effective in vitro activation of naive CD8 T cells requires three signals. TCR and CD28 signals are sufficient to stimulate proliferation, but a signal provided by IL-12 or Type I IFN (IFN1/2) is required to stimulate differentiation. During the current granting period peptide immunization, tumor and pathogen models have provided a great deal of evidence in support of this three signal model. We have also found that IL-12 and IFN1 induce a gene regulation program that results in up-or down- regulation of about 350 genes, superimposed on the regulation that occurs in response to TCR and CD28 signals. A number of transcription factors are regulated by IL-12/IFN1, including T-bet and eomesodermin (Eomes). In addition, IL- 12/FN1 upregulate expression of B lymphocyte-induced maturation protein-1 (Blimp-1), the 'master regulator' of terminal B cell differentiation, and downregulate a number of other transcription repressors, including Bcl-6. Preliminary results strongly support the hypotheses that: 1) CD8 T cells initiate the differentiation program in response to 2 signals, but in the absence of a third signal programming is terminated as a result of upregulated expression of multiple transcriptional repressors, and 2) Blimp-1, and its counter-repressor Bcl-6, play central roles in signal 3-dependent regulation of gene expression in Ag-activated CD8 T cells, with Blimp-1 acting to repress expression of transcriptional repressors that terminate differentiation. Thus, Blimp-1 may act as a 'master regulator' in CD8 T cell differentiation and Bcl-6 may have a role in regulating development of memory, somewhat analogous to their roles in formation of plasma cells and memory B cells. Two Specific Aims are proposed to test these and additional hypotheses: Specific Aim 1. To determine the role of Blimp-1 in regulating gene expression for signal 3-dependent CD8 T cell programming. The roles of Blimp-1 in regulating IL-12/IFN1-dependent changes in gene expression will be examined in both in vitro and in vivo models. We will focus on roles for Blimp-1 in regulating Bcl-6 and other transcription repressors, and the positive transcription factors T-bet and Eomes. Specific Aim 2. To determine the roles of Blimp-1/Bcl-6 in regulating gene expression for CD8 T cell differentiation and memory formation. Bcl-6 is likely to have a key role in regulating CD8 T cell differentiation, at least in part through its role as a counter-repressor of Blimp-1, and this will be examined. Furthermore, the balance of Blimp-1/Bcl-6 expression may regulate the transition to memory, and may determine whether an activated CD8 T cell becomes an effector memory cell or a central memory cell. Several hypotheses regarding Blimp-1/Bcl-6 roles in this transition will be tested. We anticipate that the planned experiments will provide significant new insights into the molecular regulation of the CD8 T cell differentiation program, and that the results will have implications for the design of prophylactic and therapeutic vaccines that target CD8 T cell responses.